The goals of this study are to investigate the mechanisms by which soft metals, cystine import, and cysteine accumulation can perturb the fitness of Escherichia coli. These three distinct stresses are connected by the central involvement of cysteine chemistry. Soft metals are present in many habitats, where they comprise a threat to bacteria and higher organisms alike. Their toxicity is underscored by the distribution throughout the biota of dedicated detoxification systems that bind and export them. Some of these metals- notably silver and mercury-have a long history of being used as antibiotics. Nevertheless, we have little knowledge of how they actually damage cells. In recent published work we demonstrated that copper poisons E. coli primarily by inactivating Fe/S-dependent dehydratases. It does so by binding the cysteine residues that coordinate the catalytic iron-sulfur clusters of these enzymes, thereby displacing the iron atoms. Preliminary data demonstrate that silver, mercury, cadmium, and zinc have this effect in vitro, too. This study (Aim 1) will test whether these soft metals exert their toxicity through this mechanism in vivo. It will also determine whether soft metals similarly displace iron from mononuclear enzymes, which employ coordinating groups-often including cysteine-that prefer metals other than iron. Aim 2 focuses upon a separate sulfur problem: the risk of disulfide stress when cystine is imported from aerobic environments. Disulfide stress has conventionally been thought to arise from reactive oxygen species, but recent data do not support this idea. However, disulfide stress is a real risk when cells rapidly import disulfide compounds, such as cystine. In principle such an event would seem likely to propagate disulfide bonds to cytoplasmic proteins. Our study of the high-flux transporter suggests that cystine import is linked to reduction, a tacti that would avoid the release of this disulfide into the cytoplasm. In Aim 2 this model will be rigorously tested. Aim 3 addresses the consequence of rapid cystine import: the excessive accumulation of cysteine, which is toxic in its own right. Our data reveals that E. coli deals with this problem by pumping the cysteine back out of the cell. This investigation will test three plausible mechanisms of cysteine toxicity, and it will identify the exporter(s) that averts it. Collectively this work will illuminate chemical problems that arise from the redox activity and metal affinity of cysteine, as well as the strategies that cells have acquired to protect themselve from it. All three of these sulfur-focused stresses-soft-metal exposure, disulfide import, and cysteine accumulation-occur under conditions that are likely to exist in natural habitats. PUBLIC HEALTH RELEVANCE: The bacteriocidal actions of soft metals have long been exploited, from the administration of mercury as an antibiotic to the coating of catheters with silver. This study seeks to illuminate how these metals exert their effects-which may enable workers to refine how they are employed. We also will examine how E. coli strives to minimize the intracellular concentrations of both disulfide and free-cysteine species. The loss of this control comprises a type of oxidative stress that is believed to contribute to a wide variety of human pathologies.